1. Field of the Invention
The present invention relates to a method of obtaining proteins from bacterial cells. More particularly, the present invention relates to a method of releasing periplasmic proteins from bacterial cells.
2. The Prior Art
Several methods have been used to release periplasmic proteins from gram-negative bacteria. Treating cells with ethlyenediaminetetraacetate and lysozyme causes the release of periplasmic proteins and the formation of spheroplasts. Another procedure is that of osmotic shock. In this method, bacteria are treated with 40% sucrose, 0.05 molar TRIS-Cl pH 7.8, and 0.002 molar EDTA, followed by cold shock with distilled water.
Solvents have been used to good advantage in permeabilizing and partially lysing bacterial cells. Toluene and phenethyl alcohol have been used to permeabilize cells in order to perform in situ enzyme assays. In order to select for mutants of L-arabinose binding protein, Hogg, J. Bacteriol. 105:604, 1971 used a mixture of chloroform-toluene 1:1 to partially lyse cells on culture plates. However, these prior art methods involve several steps and extensive manipulations which require, inter alia, concentration of cells out of the culture medium, exposing them to buffer solution, then to high concentration of sucrose in the presence of an ion chelator, then again concentrating the cells and suspending them in distilled water and then removing the cells from the final medium, etc. Not only these lengthy manipulative steps are cumbersome but the protein solution so obtained as a final product is also very dilute. This involves a further step of concentrating the dilute protein before it can be used for purification of individual protein components. Such lengthy and unwieldy prior art processes are simply not amenable to large scale screening of periplasmic proteins from multiple batches.
The applicants have now discovered an improved and simple procedure for extracting periplasmic proteins requiring a minimum of manipulations carried out in a comparatively short period of time to obtain a relatively pure periplasmic fraction. An additional advantage of the inventive procedure of the present invention is that it can be used in both small and large scale preparations including fermenter size cultures. Furthermore, prior art osmotic shock process is neither convenient nor efficient for application to a small volume e.g., 2 ml, of cell culture.